Method and Device for the Collection and Isolation of Nucleic Acid

ABSTRACT

A device for collecting and preserving nucleic acids in a sample, the device comprising: a) a support; b) one or more than one sample zone in the support for loading the sample onto the device; and c) a composition comprising i) one or more than one absorbent, and ii) one or more than one stabilizer; where the one or more than one sample zone on the support comprises a recess or space within the support extending from the top surface toward, but not through, the bottom surface, or comprises a space within the support and the composition is retained within the sample zone. A method for collecting and preserving nucleic acids in a sample, the method comprising a) providing a device for collecting and preserving nucleic acids in a sample according to the present invention; b) providing a sample potentially comprising one or more than one nucleic acid; and c) applying part or all of the sample to one or more than one of the sample zones on the device. A method of detecting and quantifying nucleic acids in a sample, the method comprising a) collecting and preserving nucleic acids in the sample according to a method of the present invention; b) removing the absorbent with sample from the sample zones of the device; and c) detecting, or detecting and quantifying the nucleic acids.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication 60/709,869, titled “A Method for the Collection andIsolation of mRNA,” filed Aug. 19, 2005; the contents of which areincorporated in this disclosure by reference in their entirety.

BACKGROUND

Minute amounts of mRNA can be detected and quantified from a few cellsusing highly sensitive, gene specific methods, such as for exampleqRT-PCR combined with general methods for the amplification of mRNA.mRNA to be detected and quantified is often isolated from whole bloodfor many reasons, including in studies of human disease.Disadvantageously, however, special collection techniques, resources andsupplies are required for collecting the relatively large volumes ofwhole blood needed for isolating mRNA from the whole blood, before themRNA is detected and quantified. For example, presently available RNAblood collection tubes can only be used with certain isolation methods,permit storage of the sample for a very limited time at roomtemperature, and sometimes alter the quality of RNA obtained. Further,presently available RNA blood collection tubes can be inconvenient touse in large multi-subject studies.

Therefore, there remains a need of a device for collecting andpreserving nucleic acids in a sample that are not associated with thedisadvantages of presently available RNA blood collection tubes.Further, there remains a need of a method for collecting and preservingnucleic acids in a biological sample that are not associated with thesedisadvantages.

SUMMARY

According to one embodiment of the present invention, there is provideda device for collecting and preserving nucleic acids in a sample. Thedevice comprises: a) a support comprising a top surface, an opposingbottom surface, and a lateral edge surrounding the top surface and thebottom surface; b) one or more than one sample zone in the support forloading the sample onto the device; and c) a composition comprising i)one or more than one absorbent, and ii) one or more than one stabilizer;where the one or more than one sample zone on the support comprises arecess or space within the support extending from the top surfacetoward, but not through, the bottom surface, or comprises a space withinthe support extending from the top surface completely through the bottomsurface, and where the composition is retained within the sample zone.In one embodiment, the support comprises a hydrophobic material. Inanother embodiment, the support comprises a material selected from thegroup consisting of plasticized cardboard, polyacetate, polycarbonateand polypropylene. In another embodiment, the device further comprises ashape selected from the group consisting of an oval, a circle, arectangle, a rectangle with rounded corners, a square, a square withrounded corners, a triangle and a triangle with rounded corners. Inanother embodiment, the one or more than one sample zone comprises aplurality of sample zones comprising between 2 and 1000 sample zones. Inanother embodiment, the one or more than one sample zone comprises aplurality of sample zones comprising between 2 and 500 sample zones. Inanother embodiment, the one or more than one sample zone comprises aplurality of sample zones comprising between 20 and 200 sample zones. Inanother embodiment, the device comprises a plurality of sample zones,and the shape of each sample zone is identical to every other samplezone. In another embodiment, the device comprises a plurality of samplezones, and the shape of at least one sample zone is different that theshape of at least one other sample zone. In another embodiment, theshape of at least one of the one or more than one sample zone, as viewedfrom the top surface, comprises a shape selected from the groupconsisting of an oval, a circle, a rectangle, a square and a triangle.In another embodiment, the composition filling the one or more than onesample zone is in a solid state. In another embodiment, the one or morethan one absorbent comprises a polymeric material in either fibrous orparticulate form. In another embodiment, the one or more than oneabsorbent comprises a hydrophilic material. In another embodiment, theabsorbent consists of a single material. In another embodiment, theabsorbent comprises a plurality of materials. In another embodiment, theone or more than one absorbent is selected from the group consisting ofcarbon, cellulose acetate, cellulose beads, cellulose fibers, celluloseparticles, dextran fibers, dextran particles, diatomaceous earth,hydroxyapatite, nitrocellulose, nylon, polyesters, polyethylene andsilica. In another embodiment, the one or more than one stabilizercomprises a substance selected from the group consisting of a dodecylsulfate as its sodium, a lithium salt, an anionic salt, a potassiumsalt, cetyl pyridinium hydrochloride, guinidinium hydrochloride,guinidinium thiocyanate, lithium sulphate and potassium sulphate. Inanother embodiment, the stabilizer comprises a buffer selected from thegroup consisting of MOPS and TRIS. In another embodiment, the stabilizercomprises an antioxidant selected from the group consisting of ascorbicacid, disodium ethylene tetra acetic acid (Na₂ EDTA), dithiothreitol,ethyl parabens and methyl parabens. In another embodiment, thestabilizer comprises a substance that inhibits nucleases, such as forexample ribonucleases, where the substance is selected from the groupconsisting of aurine tricarboxylic acid, one or more than oneguinidinium salts, placental ribonuclease inhibitor and vanadylcomplexes. In another embodiment, the sample zones further comprise adepression in the surface of each sample zone as viewed from the topsurface. In another embodiment, the device further comprises a handle.In another embodiment, the handle is a loop.

According to another embodiment of the present invention, there isprovided a method of making a device for collecting and preservingnucleic acids in a sample according to the present invention. In oneembodiment, the method comprises: a) providing the support; b) providingthe one or more than one absorbent, and the one or more than onestabilizer; and c) filling the one or more than one sample zone in thesupport with the one or more than one absorbent and the one or more thanone stabilizer by: i) filling the one or more than one sample zone inthe support with the one or more than one absorbent, and then applyingthe one or more than one stabilizer to the absorbent in each of thesample zones; or ii) producing a composition comprising the one or morethan one absorbent and the one or more than one stabilizer, and thenfilling the one or more than one sample zone in the support with thecomposition. In one embodiment, the composition is produced by combiningthe one or more than one absorbent and the one or more than onestabilizer in an aqueous solution to produce a paste or slurry. Inanother embodiment, the method further comprises removing contaminatingnucleic acids from the one or more than one absorbent and one or morethan one stabilizer. In another embodiment, the method further comprisestreating the absorbent with a wetting agent. In another embodiment, themethod further comprises removing any excess absorbent, or excesscomposition on the device but not in a sample zone. In anotherembodiment, the method further comprises further comprising drying theabsorbent or the composition in the one or more than one sample zone.

According to another embodiment of the present invention, there isprovided a method for collecting and preserving nucleic acids in asample. The method comprises: a) providing a device according to thepresent invention; b) providing a sample potentially comprising one ormore than one nucleic acid; and c) applying part or all of the sample toone or more than one of the sample zones on the device. In oneembodiment, the sample is a biological sample. In another embodiment,where the sample is selected from the group consisting of a cellculture, a cell suspension, biopsy aspirates, bone marrow, cerebrospinalfluid, potable water, plasma, serum, urine and whole blood. In anotherembodiment, the nucleic acids in the sample are selected from the groupconsisting DNA and RNA. In another embodiment, the nucleic acids in thesample are selected from the group consisting of mRNA, miRNA andmitochondrial RNA. In another embodiment, the nucleic acids in thesample are selected from the group consisting of genomic DNA andmitochondrial DNA. In another embodiment, the sample provided is from aeukaryote. In another embodiment, the sample provided is from a primate.In another embodiment, the sample provided is from a human. In anotherembodiment, the method further comprises drying the applied sample. Inanother embodiment, the device provided comprises depressions in thesample zones, and applying the sample to the one or more than one samplezones comprises applying a predetermined amount of sample based on thevolume of the depression. In another embodiment, the method furthercomprises collecting the sample into a vessel before applying the sampleto the one or more than one sample zones. In another embodiment, themethod further comprises storing the device for a time between 1 minuteand 10 years. In another embodiment, the method further comprisesstoring the device for a time between 1 day and 1 years. In anotherembodiment, the method further comprises storing the device for a timebetween 1 day and 100 days. In another embodiment, the method furthercomprises sealing the device in a protective container before beingstored.

According to another embodiment of the present invention, there isprovided a method of detecting and quantifying nucleic acids in asample. The method comprises: a) collecting and preserving nucleic acidsin the sample according to the method of the present invention; b)removing the absorbent with sample from the sample zones of the device;and c) detecting, or detecting and quantifying the nucleic acids. In oneembodiment, the method further comprises performing a technique selectedfrom the group consisting of PCR, RT-PCR, and quantitative RT-PCR.

DRAWINGS

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures where:

FIG. 1 is a top perspective schematic view of a device according to thepresent invention;

FIG. 2 is a cross-sectional, lateral perspective schematic view of thedevice according to the present invention as shown in FIG. 1 taken alongline 2-2;

FIG. 3 is a bar chart showing the qRT-PCR results of varioushousekeeping genes at various intervals over a 117-day period using thepresent method compared to control;

FIG. 4 is a graph showing real-time PCR standard curves of the 18S geneusing 10-fold serial dilutions of commercially available cDNA; and

FIG. 5 is a graph showing 18S gene results for test zones obtained day 1through day 117.

DESCRIPTION

According to one embodiment of the present invention, there is provideda device for collecting and preserving nucleic acids in a sample. Thedevice comprises a support and at least one sample zone on the supportfor loading the stabilizer onto the device. In a preferred embodiment,the nucleic acids in the stabilizer are mRNA. According to anotherembodiment of the present invention, there is provided a method ofmaking a device for collecting and preserving nucleic acids in a sample.In one embodiment, the method comprises providing an absorbent and thenfilling one or more sample zone in a support with the absorbent, andthen applying one or more than one stabilizer to the absorbent. Inanother embodiment, the method comprises forming a composition bycombining one or more than one absorbent and one or more than onestabilizer, and then filling one or more sample zone in a support withthe composition. According to another embodiment of the presentinvention, there is provided a method for collecting and preservingnucleic acids in a sample. In one embodiment, the sample is a biologicalsample. In one embodiment, the method comprises providing a device forcollecting and preserving nucleic acids in a sample according to thepresent invention. In a preferred embodiment, the nucleic acids in thebiological sample are mRNA. According to another embodiment of thepresent invention, there is provided a method of detecting andquantifying nucleic acids in a sample. In one embodiment, the sample isa biological sample. In another embodiment, the method comprisescollecting and preserving nucleic acids in a sample according to amethod of the present invention. In a preferred embodiment, the nucleicacids in the sample are mRNA. The device and methods will now bedisclosed in greater detail.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised” are not intended to exclude other additives, components,integers or steps.

All dimensions specified in this disclosure are by way of example onlyand are not intended to be limiting. Further, the proportions shown inthese Figures are not necessarily to scale. As will be understood bythose with skill in the art with reference to this disclosure, theactual dimensions of any device or part of a device disclosed in thisdisclosure will be determined by its intended use.

As used in this disclosure, except where the context requires otherwise,the method steps disclosed and shown are not intended to be limiting norare they intended to indicate that each step is essential to the methodor that each step must occur in the order disclosed.

According to one embodiment of the present invention, there is provideda device for collecting and preserving nucleic acids in a sample. In oneembodiment, the sample is a biological sample. Referring now to FIG. 1and FIG. 2, there are shown, respectively, a top perspective schematicview of a device according to the present invention (FIG. 1); and across-sectional, lateral perspective schematic view of the deviceaccording to the present invention as shown in FIG. 1 taken along line2-2 (FIG. 2). As can be seen, the device 10 comprises a support 12comprising a top surface 14, an opposing bottom surface 16, and alateral edge 18 surrounding the top surface 14 and the bottom surface16. The support 12 further comprises one or more than one sample zone 20in the support 12 for loading the sample onto the device 10.

The shape of the device 10 is determined according to the intended useof the device 10, as will be understood by those with skill in the artwith reference to this disclosure. In one embodiment, for example, thetop surface 14 of the device 10 comprises a shape selected from thegroup consisting of an oval, a circle, a rectangle, a rectangle withrounded corners, a square, a square with rounded corners, a triangle anda triangle with rounded corners. In a preferred embodiment, the shape ofthe top surface 14 of the device 10 is a rectangle comprising a longside 22 and a short side 24 as shown in FIG. 1.

The dimensions of the device 10 are also selected according to theintended use of the device 10, as will be understood by those with skillin the art with reference to this disclosure. In a preferred embodiment,the dimensions of the device 10 are selected so that the device 10 iseasily portable by hand carrying and easily storable in typical drawersor cabinets in a research lab. For example, in one embodiment, thedevice 10 is a rectangle with rounded corners comprising a long side 22and a short side 24 as shown in FIG. 1, and the long side 22 is between4 cm and 50 cm. In another embodiment, the device 10 is a rectangle withrounded corners comprising a long side 22 and a short side 24 as shownin FIG. 1, and the long side 22 is between 4 cm and 25 cm. In anotherembodiment, the device 10 is a rectangle with rounded corners comprisinga long side 22 and a short side 24 as shown in FIG. 1, and the long side22 is between 4 cm and 10 cm. In another embodiment, the device 10 is arectangle with rounded corners comprising a long side 22 and a shortside 24 as shown in FIG. 1, and the short side 24 is between 1 cm and 10cm. In another embodiment, the device 10 is a rectangle with roundedcorners comprising a long side 22 and a short side 24 as shown in FIG.1, and the short side 24 is between 1 cm and 5 cm. In anotherembodiment, the device 10 is a rectangle with rounded corners comprisinga long side 22 and a short side 24 as shown in FIG. 1, and the shortside 24 is between 1 cm and 2 cm. In another embodiment, the device 10is a rectangle with rounded corners comprising a thickness 26 as shownin FIG. 2, and the thickness 26 is between 1 mm and 10 mm. In anotherembodiment, the device 10 is a rectangle with rounded corners comprisinga thickness 26 as shown in FIG. 2, and the thickness 26 is between 1 mmand 5 mm. In another embodiment, the device 10 is a rectangle withrounded corners comprising a thickness 26 as shown in FIG. 2, and thethickness 26 is between 1 mm and 2 mm.

In one embodiment, the support 12 of the device 10 comprises a materialthat is inert with respect to nucleic acids. In a preferred embodiment,the support 12 comprises a hydrophobic material. In a preferredembodiment, the support 12 comprises a material selected from the groupconsisting of plasticized cardboard, polyacetate, polycarbonate andpolypropylene.

As disclosed above, the support 12 further comprises one or more thanone sample zone 20 in the support 12 for loading the sample onto thedevice 10. In one embodiment, the one or more than one sample zone 20 onthe support 12 comprises a recess or space within the support 12extending from the top surface 14 toward, but not through, the bottomsurface 16. In a particularly preferred embodiment, the one or more thanone sample zone 20 on the support 12 comprises a space within thesupport 12 extending from the top surface 14 completely through thebottom surface 16.

In one embodiment, the one or more than one sample zone 20 comprises aplurality of sample zones 20 comprising between 2 and 1000 sample zones20. In another embodiment, the one or more than one sample zone 20comprises a plurality of sample zones 20 comprising between 2 and 500sample zones 20. In another embodiment, the one or more than one samplezone 20 comprises a plurality of sample zones 20 comprising between 20and 200 sample zones 20.

In one embodiment, the device comprises a plurality of sample zones 20,and the shape of each sample zone 20 is identical to every other samplezone 20. In one embodiment, the device comprises a plurality of samplezones 20, and the shape of at least one sample zone 20 is different thatthe shape of at least one other sample zone 20. In one embodiment, theshape of at least one of the one or more than one sample zone 20, asviewed from the top surface 14, comprises a shape selected from thegroup consisting of an oval, a circle, a rectangle, a square and atriangle.

The dimensions of the one or more than one sample zone 20 depend on thedimensions of the support 12, as will be understood by those with skillin the art with reference to this disclosure. By way of example only, inone embodiment, the one or more than one sample zone 20 comprises arecess or space within the support 12 extending from the top surface 14toward, but not through, the bottom surface 16 maximally between 20% and90% of the thickness 26 of the support 12. In another embodiment, theone or more than one sample zone 20 comprises a recess or space withinthe support 12 extending from the top surface 14 toward, but notthrough, the bottom surface 16 maximally between 30% and 80% of thethickness 26 of the support 12. In another embodiment, the one or morethan one sample zone 20 comprises a recess or space within the support12 extending from the top surface 14 toward, but not through, the bottomsurface 16 maximally between 25% and 75% of the thickness 26 of thesupport 12. By way of example only, in one embodiment, the one or morethan one sample zone 20 comprises a recess or space within the support12 extending from the top surface 14 toward, but not through, the bottomsurface 16 maximally between 20% and 90% of the thickness 26 of thesupport 12.

By way of example only, in one embodiment, each sample zone 20 is roundas viewed form the top surface 14, and each sample zone 20 has adiameter between 1 mm and 20 mm. In another embodiment, each sample zone20 is round as viewed form the top surface 14, and each sample zone 20has a diameter between 1 mm and 10 mm. In another embodiment, eachsample zone 20 is round as viewed form the top surface 14, and eachsample zone 20 has a diameter between 1 mm and 5 mm. Further by way ofexample only, in one embodiment, each sample zone 20 extends from thetop surface 14 toward, but not through, the bottom surface 16 maximallybetween 0.5 mm and 10 mm. In another embodiment, each sample zone 20extends from the top surface 14 toward, but not through, the bottomsurface 16 maximally between 1 mm and 5 mm. In another embodiment, eachsample zone 20 extends from the top surface 14 toward, but not through,the bottom surface 16 maximally between 1 mm and 2 mm.

The one or more than one sample zone 20 further comprises a composition28 comprising a) one or more than one absorbent, and b) one or more thanone stabilizer. In a preferred embodiment, the composition 28 fillingthe one or more than one sample zone 20 is in a solid state.

The one or more than one absorbent functions to absorb the sample ontothe sample zone 20 without irreversibly binding the nucleic acids in thesample. The absorbent comprises a material that can be dried afterapplication to the sample zone 20 without powdering or otherwisebreaking or detaching from the surface of the sample zone 20, withoutthe application of an external force to cause detachment of thecomposition from the walls of the sample zone 20.

In one embodiment, the one or more than one absorbent comprises apolymeric material in either fibrous or particulate form. In anotherembodiment, the one or more than one absorbent comprises a hydrophilicmaterial. In one embodiment, the absorbent consists of a singlematerial. In another embodiment, the absorbent comprises a plurality ofmaterials. In another embodiment, the one or more than one absorbent isselected from the group consisting of carbon, cellulose acetate,cellulose beads, cellulose fibers, cellulose particles, dextran fibers,dextran particles, diatomatious earth, hydroxyapatite, nitrocellulose,nylon, polyesters, polyethylene and silica. In a preferred embodiment,the absorbent comprises a material which is substantially free (that ismore than 99.9%) of heavy metals or other constituents which causenucleic acids to breakdown. In another preferred embodiment, theabsorbent comprises a material which is substantially free (that is morethan 99.9%) of nucleic acids which can arise from the use of materialsproduced from fermentation.

The one or more than one stabilizer functions to diminish or preventbreakdown of the nucleic acid in the sample, such as for example, bydenaturing proteins that can inactivate polynucleotides, that cansequester polynucleotides, and by facilitating the disassociation ofnucleases from nucleic acids, thereby freeing the nucleic acids forminterference by such nucleases.

In a preferred embodiment, the one or more than one stabilizer performsone or more than one function selected from the group consisting ofantioxidation, buffering, cell lysis, chelation of metal cofactors suchas calcium or magnesium, nuclease inhibition, and protection of nucleicacids from oxidative degradation or the action of microbialcontaminants. When used as a buffer, the stabilizing should maintain pHbetween 6.0 and 8.0, preferably between pH 6.2 and 7.0, and mostpreferably between 6.4 and 6.8. In another embodiment, the one or morethan one stabilizer comprises a substance selected from the groupconsisting of a dodecyl sulfate as its sodium, a lithium salt, ananionic salt, a potassium salt, cetyl pyridinium hydrochloride,guanidinium hydrochloride, guanidinium thiocyanate, lithium sulphate andpotassium sulphate. In one embodiment, the stabilizer comprises a bufferselected from the group consisting of MOPS and TRIS. In anotherembodiment, the stabilizer comprises an antioxidant that decreases orprevents oxidative degradation of the nucleic acids selected from thegroup consisting of ascorbic acid, disodium ethylene tetra acetic acid(Na₂ EDTA), dithiothreitol, ethyl parabens and methyl parabens. Inanother embodiment, the stabilizer comprises a substance that inhibitsnucleases, such as for example ribonucleases, where the substance isselected from the group consisting of aurine tricarboxylic acid, one ormore than one guanidinium salts, placental ribonuclease inhibitor andvanadyl complexes. In one embodiment, the amount of the one or more thanone stabilizers is sufficient to preserve and stabilize the nucleicacids in the sample, as will be understood by those with skill in theart with reference to this disclosure.

As will be understood by those with skill in the art with reference tothis disclosure, the absolute and relative amounts of the absorbent andstabilizer present in each sample zone 20 is selected to be sufficientfor the purposes disclosed in this disclosure.

In one embodiment, the composition further comprises an additive thatfunctions to bind the absorbent into a solid phase or to the walls ofthe sample zone 20, or both to bind the absorbent into a solid phase andto the walls of the sample zone 20. In one embodiment, the additive isselected from the group consisting of albumin, gelatin, polyvinylalcohol, starch, sucrose, trehalose, polyacrylamide, and polyethyleneglycol.

In one embodiment, the sample zones 20 further comprise a depression 30in the surface of each sample zone 20 as viewed from the top surface 14.The depression 30 is configured to accept a predetermined sample volume.By way of example only, in one embodiment, the depression 30 comprises avolume, as measured from the plane of the top surface 14 to the surfaceof the composition within the sample zone 20 of between 1 and 100 μl. Inanother embodiment, the depression 30 comprises a volume, as measuredfrom the plane of the top surface 14 to the surface of the compositionwithin the sample zone 20 of between 2 and 50 μl. In another embodiment,the depression 30 comprises a volume, as measured from the plane of thetop surface 14 to the surface of the composition within the sample zone20 of between 5 and 20 μl.

In one embodiment, the device 10 further comprises a handle 32 to assistin manually grasping and manipulating the device. The handle 32 can beany suitable size and shape for the intended purpose as will beunderstood by those with skill in the art with reference to thisdisclosure. In one embodiment, the handle 32 comprises a loop as shownto permit storage of a plurality of devices 10 by threading the openportion of the loop onto a strut or peg.

According to another embodiment of the present invention, there isprovided a method of making a device for collecting and preservingnucleic acids in a sample. In one embodiment, the device made accordingto this method is useful for collecting and preserving mRNA in thestabilizer. In a preferred embodiment, the device made according to thismethod is a device 10 according to the present invention.

In one embodiment, the method comprises, first, providing a support,such as a support 12 comprising one or more than one sample zone 20, asdisclosed in connection with the device 10 according to the presentinvention. In a preferred embodiment, the support is produced usinginjection molding to form a plastic material into the desired shape,however, any suitable technique can be used, as will be understood bythose with skill in the art with reference to this disclosure. In oneembodiment, the method comprises forming one or more than one samplezone in the support by drilling or by an equivalent technique, as willbe understood by those with skill in the art with reference to thisdisclosure.

In a preferred embodiment, the shape and dimensions of the support arein accordance with the shape and dimensions disclosed in connection withthe support 12 for the device 10.

Next, the method comprises providing one or more than one absorbent, andone or more than one stabilizer in accordance with the absorbents andstabilizers as disclosed in connection with the device 10. In oneembodiment, the method comprises filling the one or more than one samplezone in the support with the one or more than one absorbent, and thenapplying the one or more than one stabilizer to the absorbent in each ofthe sample zones. In an alternate embodiment, the method comprisesproducing a composition comprising the one or more than one absorbentand the one or more than one stabilizer, and then filling the one ormore than one sample zone in the support with the composition. In apreferred embodiment, the composition provided is a composition 28, asdisclosed in connection with the device 10 according to the presentinvention, comprising a) one or more than one absorbent, and b) one ormore than one stabilizer. In one embodiment, the composition is producedby combining the one or more than one absorbent and the one or more thanone stabilizer in an aqueous solution to produce a paste or slurry.

By way of example, an absorbent was produced from 20 micron celluloseSigmacell Type 101 (Sigma, St. Louis, Mo. US) particles suspended inwater and formed into a paste. The paste was then applied to each of 10sample zones comprising round through holes present in a rectangularpolymer support, and the support with the absorbent filled sample zoneswas allowed to air dry at 50° C. for 2 hours. A stabilizer comprising anaqueous solution of 1% Sodium Dodecyl Sulfate, 10 mM EDTA, 10 mM MOPS,500 mM lithium chloride and 5 mM ammonium salt of aurine tricarboxylicacid, pH 6.8 (all from Sigma) was prepared, and the stabilizer wasapplied to the absorbent in each of five of the ten sample zones. Thedevice was then allowed to dry for 2 hours at 50° C.

In another embodiment, the method comprises placing less of theabsorbent, or less of the composition, in each of the one or more thanone of the sample zones than is needed to fill the sample zone, therebyleaving a depression, as disclosed in connection with the device 10.

In a preferred embodiment, the method further comprises removingcontaminating nucleic acids from the one or more than one absorbent andone or more than one stabilizer using techniques as will be understoodby those with skill in the art with reference to this disclosure, suchas for example, the addition of one or more than one nuclease to the oneor more than one absorbent and one or more than one stabilizer, allowingthe one or more than one nuclease to degrade any contaminating nucleicacid, and then removing, inactivating, or both removing andinactivating, the one or more than one nuclease following their use todegrade any contaminating nucleic acids.

In another embodiment, the method further comprises treating theabsorbent with a wetting agent to render a substantially hydrophobicmaterial, such as nylon, suitable for use in the composition. In oneembodiment, the wetting agent is selected from the group consisting of asurfactant, an ionic detergent and a nonionic detergent.

In a preferred embodiment, the method further comprises removing anyexcess absorbent, or excess composition on the device but not in asample zone, such as for example by wiping the excess absorbent orexcess composition from the top surface and the bottom surface of thedevice.

In one embodiment, the method further comprises drying the absorbent orthe composition in the one or more than one sample zone. Drying can beaccomplished passively by air drying at room temperature, or can beaccomplished by the application of heat, the application of a vacuum, orthe application of both heat and a vacuum, as will be understood bythose with skill in the art with reference to this disclosure. Dryingfunctions to remove bulk water or other solvents from the absorbent orfrom the composition.

In another embodiment, the method comprises filling the one or more thanone sample zone in the support with the one or more than one absorbent,and then applying the one or more than one stabilizer to the absorbentin each of the sample zones, and the method further comprises drying theabsorbent with the applied stabilizer. Drying can be accomplishedpassively by air drying at room temperature, or can be accomplished bythe application of heat, the application of a vacuum, or the applicationof both heat and a vacuum, as will be understood by those with skill inthe art with reference to this disclosure. Drying functions to removebulk water or other solvents from the absorbent or from the composition.

According to another embodiment of the present invention, there isprovided a method for collecting and preserving nucleic acids in asample. In one embodiment, the method comprises providing a device forcollecting and preserving nucleic acids in a sample according to thepresent invention. In a preferred embodiment, the device is a device 10according to the present invention.

Next, the method comprises providing a sample potentially comprising oneor more than one nucleic acid. In one embodiment, the sample is abiological sample. In a preferred embodiment, the sample is selectedfrom the group consisting of a cell culture, a cell suspension, biopsyaspirates, bone marrow, cerebrospinal fluid, potable water, plasma,serum, urine and whole blood. In another embodiment, the nucleic acidsin the sample are selected from the group consisting DNA and RNA. In apreferred embodiment, the nucleic acids in the sample are selected fromthe group consisting of mRNA, miRNA and mitochondrial RNA. In apreferred embodiment, the nucleic acids in the sample are selected fromthe group consisting of genomic DNA and mitochondrial DNA.

In one embodiment, the sample provided is from a eukaryote. In anotherembodiment, the sample provided is from a primate. In anotherembodiment, the sample provided is from a human. In a preferredembodiment, providing the sample comprises obtaining whole blood from ahuman by a finger stick.

Next, part or all of the sample is applied to one or more than one ofthe sample zones on the device. In a preferred embodiment, the appliedsample is then allowed to dry while protected from contamination, underconditions suitable for the nucleic acid being collected and preserved,as will be understood by those with skill in the art with reference tothis disclosure. For example, drying can be accomplished passively byair drying at room temperature, or can be accomplished by theapplication of heat, the application of a vacuum, or the application ofboth heat and a vacuum, as will be understood by those with skill in theart with reference to this disclosure. Drying functions to remove bullwater or other solvents from the absorbent or from the composition.

In another embodiment, between 1 μl and 1000 μl of sample is applied toeach sample zone. In another embodiment, between 1 μl and 100 μl ofsample is applied to each sample zone. In another embodiment, between 1μl and 10 μl of sample is applied to each sample zone.

In a preferred embodiment, the device provided comprises depressions inthe sample zones, and applying the sample to the one or more than onesample zones comprises applying a predetermined amount of sample basedon the volume of the depression.

In one embodiment, the method further comprises collecting the sampleinto a vessel before applying the sample to the one or more than onesample zones. Collecting the sample into a vessel before applying thesample to the sample zone assists in assuring that a predeterminedamount of the sample is applied, as will be understood by those withskill in the art with reference to this disclosure. In one embodiment,the vessel is selected from the group consisting of a capillary tube anda pipette tip collection tube.

Once, the sample zones are dry, the device with the nucleic acids in thesample zones can be stored for future use. In one embodiment, the deviceis stored for a time between 1 minute and 10 years. In anotherembodiment, the device is stored for a time between 1 day and 1 year. Inanother embodiment, the device is stored for a time between 1 day and100 days.

In another embodiment, the device is sealed in a protective containerbefore being stored. In one embodiment, the protective container isselected from the group consisting of a tube and a box.

In one embodiment, the device is stored at ambient temperature. Inanother embodiment, the device is stored at a temperature between −20°C. and −80° C. In another embodiment, the device is stored at atemperature between 0° C. and −100° C.

According to another embodiment of the present invention, there isprovided a method of detecting and quantifying nucleic acids in asample. In one embodiment, the method comprises, first, collecting andpreserving nucleic acids in a sample according to a method of thepresent invention.

In one embodiment, the sample is a biological sample. In a preferredembodiment, the sample is selected from the group consisting of a cellculture, a cell suspension, biopsy aspirates, bone marrow, cerebrospinalfluid, potable water, plasma, serum, urine and whole blood. In anotherembodiment, the nucleic acids in the sample are selected from the groupconsisting DNA and RNA. In a preferred embodiment, the nucleic acids inthe sample are selected from the group consisting of mRNA, miRNA andmitochondrial RNA. In a preferred embodiment, the nucleic acids in thesample are selected from the group consisting of genomic DNA andmitochondrial DNA.

Next, the method comprises removing the absorbent with sample from thesample zones of the device. In one embodiment, removing comprisespushing the absorbent with the sample through the sample zone, therebydetaching the absorbent with the sample from the device. In anotherembodiment, removing comprises scooping the absorbent with the samplefrom the sample zone, thereby detaching the absorbent with the samplefrom the device.

Then, the absorbent with the sample is processed by methods well knownin the art to obtain isolated nucleic acids such as DNA or RNA,including mRNA and micro RNAs, to remove the nucleic acids from theabsorbent.

The nucleic acids are then subject to detection and quantification usingstandard techniques, as will be understood by those with skill in theart with reference to this disclosure, such as for example,amplification methods selected from the group consisting of PCR, RT-PCR,and quantitative RT-PCR.

In one embodiment, the method comprises eluting the nucleic acids fromthe absorbent using standard techniques, as will be understood by thosewith skill in the art with reference to this disclosure. Next, thenucleic acid is isolated from the eluted material using standardtechniques, as will be understood by those with skill in the art withreference to this disclosure.

By way of example, the above steps were performed as follows. Afterapplication of 10 μl of whole blood to each sample zone, the blood wasallowed to absorb and to visibly dry. Control and test blood zones werethen punched from the device into individual tubes using a small plasticdowel for each of the successive time points. The punches for the firstthree time points were stored at −80° C. until they could be processed.The punches were each processed by adding 100 μl of the above lysispreservation buffer to each, and completely dispersing the cellulosepunches containing the applied blood into this solution. Then, 5 μl ofpoly-dT paramagnetic beads (Dynabeads, Dynal Biotech, L.L.C., BrownDeer, Wis. US) was added to the suspension of dispersed blood spots orpunches and was allowed to incubate at room temperature for 4 minuteswith repeated mixing by inversion. Paramagnetic beads were separatedfrom the mixture components by inversion of the capped tubes containingthe mixture and application of a magnet to the cap of the inverted tubefor about 2 minutes. Next, the tubes were placed upright, allowing theliquid phase to drain into the tube while the magnet applied to the capretained the mRNAs captured by the poly-dT paramagnetic beads. Each capwas then transferred to a fresh tube containing Wash Buffer A (DynalDynal Biotech). Then, the magnet was withdrawn from the cap, and thebeads and wash buffer were mixed and incubated for about 2 minutes atroom temperature. The cap transfer and incubation steps were repeated asdescribed above using 2 fresh tubes of 200 μl each of Wash Buffer B(Dynal Dynal Biotech). The mRNAs were eluted from the beads by theaddition of 10 μl 10 mM Tris-HCl pH 7.5 (Dynal Dynal Biotech) andheating to 60° C. for 5 minutes followed by centrifugation at 5000×g topellet the beads. The supernatant containing the mRNA was transferred toa fresh tube and stored at −80° C. until processing to produce cDNA andqRT-PCR by standard methods known in the art.

In one embodiment, the method further comprises quantifying the isolatednucleic acids using standard techniques, as will be understood by thosewith skill in the art with reference to this disclosure. In anotherembodiment, the method further comprises performing expression analysison the isolated nucleic acids using standard techniques, as will beunderstood by those with skill in the art with reference to thisdisclosure. In a preferred embodiment, the expression analysis isqRT-PCR.

By way of example, the method for the isolation of mRNA, and fordownstream analysis from finger-stick whole blood on an absorbent matrixaccording to the present invention was performed as follows. First, 10μl whole blood was collected using a finger stick and collection of thewhole blood into a pipette tip collection or capillary tube. Next, thewhole blood was placed on multiple absorbent matrix zones containinglysing and stabilizing agents to prevent RNA degradation.

Then, mRNA isolation was completed for each zone by eluting the samplefrom the absorbent matrix, isolation of mRNA using oligo(dT) magneticbead techniques and elution of mRNA in a stabilizing buffer. Next, theisolated mRNA was quantified using a Nanodrop® spectrophotometer(NanoDrop Technologies, Inc., Wilmington, Del. US).

Then, expression analysis was performed on the isolated mRNA by qRT-PCRusing TaqMan® gene expression assays (Applied Biosystems; Foster City,Calif. US) for a set of high, medium and low expression housekeepinggenes (18S, GAPDH, GUSb, PGK, TBP). Next, the mRNA was isolated fromsample replicate matrix zones at successive intervals extending to 117days.

Referring now to FIG. 3, FIG. 4 and FIG. 5, there are shownrespectively, a bar chart showing the qRT-PCR results of varioushousekeeping genes at various intervals over a 117-day period using thepresent method compared to control (FIG. 3); a graph showing real-timePCR standard curves of the 18S gene using 10-fold serial dilutions ofcommercially available cDNA (from Princeton BioMeditech Corporation,Princeton, N.J. US). Day 20 showed no detectable expression of the PGKgene and no time point showed detectable expression of GUSb or TBP (FIG.4); and a graph showing 18S gene results for test zones obtained day 1through day 117 (FIG. 5). As can be seen, the expression levelsdetermined from the qRT-PCR panel of housekeeping genes remainrelatively consistent across time points out to the 117-day limittested, even when stored at ambient conditions in a sealed container.The results were comparable to those from mRNA obtained fromcommercially available RNA blood tubes and isolation methods thatrequire using much larger quantities of whole blood and elaboratecollection and processing protocols.

Therefore, using the present method, the quality and quantity of mRNArequired for qRT-PCR and amplification can successfully be obtained froma finger-stick collection of whole blood on an absorbent matrix. This isparticularly useful in circumstances in which peripheral blood issuitable for expression analysis but where full scale collections arelogistically difficult and or costly.

Although the present invention has been discussed in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference totheir entirety.

1. A device for collecting and preserving nucleic acids in a sample, thedevice comprising: a) a support comprising a top surface, an opposingbottom surface, and a lateral edge surrounding the top surface and thebottom surface; b) one or more than one sample zone in the support forloading the sample onto the device; and c) a composition comprising i)one or more than one absorbent, and ii) one or more than one stabilizerin a solid state; where the one or more than one sample zone on thesupport comprises a recess or space within the support extending fromthe top surface toward, but not through, the bottom surface, orcomprises a space within the support extending from the top surfacecompletely through the bottom surface; and where the composition isretained inside the recess or space of the sample zone.
 2. The device ofclaim 1, where the support comprises a hydrophobic material.
 3. Thedevice of claim 1, where the support comprises a material selected fromthe group consisting of plasticized cardboard, polyacetate,polycarbonate and polypropylene.
 4. The device of claim 1, furthercomprising a shape selected from the group consisting of an oval, acircle, a rectangle, a rectangle with rounded corners, a square, asquare with rounded corners, a triangle and a triangle with roundedcorners.
 5. The device of claim 1, where the one or more than one samplezone comprises a plurality of sample zones comprising between 2 and 1000sample zones.
 6. The device of claim 1, where the one or more than onesample zone comprises a plurality of sample zones comprising between 2and 500 sample zones.
 7. The device of claim 1, where the one or morethan one sample zone comprises a plurality of sample zones comprisingbetween 20 and 200 sample zones.
 8. The device of claim 1, where thedevice comprises a plurality of sample zones, and the shape of eachsample zone is identical to every other sample zone.
 9. The device ofclaim 1, where the device comprises a plurality of sample zones, and theshape of at least one sample zone is different that the shape of atleast one other sample zone.
 10. The device of claim 1, where the shapeof at least one of the one or more than one sample zone, as viewed fromthe top surface, comprises a shape selected from the group consisting ofan oval, a circle, a rectangle, a square and a triangle.
 11. The deviceof claim 1, where the composition filling the one or more than onesample zone is in a solid state.
 12. The device of claim 1, where theone or more than one absorbent comprises a polymeric material in eitherfibrous or particulate form.
 13. The device of claim 1, where the one ormore than one absorbent comprises a hydrophilic material.
 14. The deviceof claim 1, where the absorbent consists of a single material.
 15. Thedevice of claim 1, where the absorbent comprises a plurality ofmaterials.
 16. The device of claim 1, where the one or more than oneabsorbent is selected from the group consisting of carbon, celluloseacetate, cellulose beads, cellulose fibers, cellulose particles, dextranfibers, dextran particles, diatomatious earth, hydroxyapatite,nitrocellulose, nylon, polyesters, polyethylene and silica.
 17. Thedevice of claim 1, where the one or more than one stabilizer comprises asubstance selected from the group consisting of a dodecyl sulfate as itssodium, a lithium salt, an anionic salt, a potassium salt, cetylpyridinium hydrochloride, guinidinium hydrochloride, guinidiniumthiocyanate, lithium sulphate and potassium sulphate.
 18. The device ofclaim 1, where the stabilizer comprises a buffer selected from the groupconsisting of MOPS and TRIS.
 19. The device of claim 1, where thestabilizer comprises an antioxidant selected from the group consistingof ascorbic acid, disodium ethylene tetra acetic acid (Na₂ EDTA),dithiothreitol, ethyl parabens and methyl parabens.
 20. The device ofclaim 1, where the stabilizer comprises a substance that inhibitsnucleases, such as for example ribonucleases, where the substance isselected from the group consisting of aurine tricarboxylic acid, one ormore than one guinidinium salts, placental ribonuclease inhibitor andvanadyl complexes.
 21. The device of claim 1, where the compositionfurther comprises an additive selected from the group consisting ofalbumin, gelatin, polyvinyl alcohol, starch, sucrose, trihalose,polyacrylamide, and polyethylene glycol.
 22. The device of claim 1,where the sample zones further comprise a depression in the surface ofeach sample zone as viewed from the top surface.
 23. The device of claim1, further comprising a handle.
 24. The device of claim 23, where thehandle is a loop.
 25. A method of making a device for collecting andpreserving nucleic acids in a sample according to claim 1, the methodcomprising: a) providing the support; b) providing the one or more thanone absorbent, and the one or more than one stabilizer; and c) fillingthe one or more than one sample zone in the support with the one or morethan one absorbent and the one or more than one stabilizer by: i)filling the one or more than one sample zone in the support with the oneor more than one absorbent, and then applying the one or more than onestabilizer to the absorbent in each of the sample zones; or ii)producing a composition comprising the one or more than one absorbentand the one or more than one stabilizer, and then filling the one ormore than one sample zone in the support with the composition.
 26. Themethod of claim 25, where the composition is produced by combining theone or more than one absorbent and the one or more than one stabilizerin an aqueous solution to produce a paste or slurry.
 27. The method ofclaim 25, further comprising removing contaminating nucleic acids fromthe one or more than one absorbent and one or more than one stabilizer.28. The method of claim 25, further comprising treating the absorbentwith a wetting agent.
 29. The method of claim 25, further comprisingremoving any excess absorbent, or excess composition on the device butnot in a sample zone.
 30. The method of claim 25, further comprisingdrying the absorbent or the composition in the one or more than onesample zone.
 31. A method for collecting and preserving nucleic acids ina sample, the method comprising: a) providing a device of claim 1; b)providing a sample potentially comprising one or more than one nucleicacid; and c) applying part or all of the sample to one or more than oneof the sample zones on the device.
 32. The method of claim 31, where thesample is a biological sample.
 33. The method of claim 32, where thesample is selected from the group consisting of a cell culture, a cellsuspension, biopsy aspirates, bone marrow, cerebrospinal fluid, potablewater, plasma, serum, urine and whole blood.
 34. The method of claim 31,where the nucleic acids in the sample are selected from the groupconsisting of DNA and RNA.
 35. The method of claim 31, where the nucleicacids in the sample are selected from the group consisting of mRNA,miRNA and mitochondrial RNA.
 36. The method of claim 31, where thenucleic acids in the sample are selected from the group consisting ofgenomic DNA and mitochondrial DNA.
 37. The method of claim 31, where thesample provided is from a eukaryote.
 38. The method of claim 31, wherethe sample provided is from a primate.
 39. The method of claim 31, wherethe sample provided is from a human.
 40. The method of claim 31, furthercomprising drying the applied sample.
 41. The method of claim 31, wherethe device provided comprises depressions in the sample zones, andapplying the sample to the one or more than one sample zones comprisesapplying a predetermined amount of sample based on the volume of thedepression.
 42. The method of claim 31, further comprising collectingthe sample into a vessel before applying the sample to the one or morethan one sample zones.
 43. The method of claim 31, further comprisingstoring the device for a time between 1 minute and 10 years.
 44. Themethod of claim 31, further comprising storing the device for a timebetween 1 day and 1 years.
 45. The method of claim 31, furthercomprising storing the device for a time between 1 day and 100 days. 46.The method of claim 31, further comprising sealing the device in aprotective container before being stored.
 47. The method of detectingand quantifying nucleic acids in a sample, the method comprising: a)collecting and preserving nucleic acids in the sample according to themethod of claim 31; b) removing the absorbent with sample from thesample zones of the device; and c) detecting, or detecting andquantifying the nucleic acids.
 48. The method of claim 47, wheredetecting, or detecting and quantifying the nucleic acids comprisesperforming a technique selected from the group consisting of PCR,RT-PCR, and quantitative RT-PCR.